07MicrobialGenetExamI
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Name_______________________________ StudentpID#__________________________ Microbial Genetics BIO 410/510 Fall Quarter 2007 Exam I 1.) Draw the structure of guanine. Indicate where the hydrogen bonds form with cytosine. (4pts) 2.) Thermosensitive mutations exist for several of the E. coli proteins associated with DNA replication. Describe the role that each of the following proteins play during semiconservative replication AND predict what would happen to replication if that protein were inactivated. EXPLAIN your rationale. (10pts) DnaB helicase: DnaG primase: DNA Ligase: Pol I: DNA Gyrase topoisomerase: Name_____________________________ page 2 3.) Escherichia coli replication machinery makes a mistake about once every 1010 times it incorporates a nucleotide. E. coli O157 is a pathogenic strain that was isolated from hamburgers at Jack in the Box restaurants in the late 1990’s. It caused severe fevers and death in several customers. Its genome contains approximately 5 * 106 basepairs. How many changes (mutations) would be expected to occur in its genome each time it replicates? Show your work. (3pts) 4.) The actual error (mutation) rate per cell division in E. coli strain O157 was measured and it was found to make a mistake about once every 106 times it incorporates a nucleotide. How many changes (mutations) occur in its genome each time it replicates? Show your work. (2pts) 5.) The mutS gene of E. coli O157 is inactivated by a mutation. MutS is part of the methyl directed mismatch repair system. Describe how DNA methylation allows replication to correct errors that were made during replication. (4pts) 6.) The E. coli origin or replication contains multiple DnaA boxes and AT-rich 13-mers. Describe what role each of these plays in the initiation of replication? (5pts) Name_____________________________ page 3 A soil bacteria that was isolated replicates in every 10 hours when grown in lab cultures. To examine whether replication occurs conservatively or semiconservatively in this bacteria, you decide to utilize a variation of the approach that Meselson-Stahl originally used to examine this question in E. coli. For your controls, you grow the bacteria in two separate culture media for several generations. C1.) One culture is grown in normal media C2.) The other culture is grown in media that contains 5-bromouracil, an analog of thymine that has a much higher buoyant density. For your experimental analysis, you inoculate a third culture of the bacteria in normal media and allow it to grow for 2 days. At this time, you then transfer the cells into media containing 5bromouracil. To examine the mode of replication, you collect a portion of the cells E1) immediately after changing the media, E2) 10 hours after you changed the media, and E3) 20 hours after you changed the media. You then lyse the cells and load the cell lysates (and DNA) from each sample in neutral CsCl gradients before centrifuging them to equilibrium. The results of your controls, C1 and C2, are shown below. 7.) On the tubes below, clearly indicate where the DNA would be expected to appear if the bacteria replicate conservatively? (5pts) 5-bromoNormal media uracil media C1 C2 0 hrs E1 10 hrs E2 8.) On the tubes below, clearly indicate where the DNA would be expected to appear if the bacteria replicate semi-conservatively? (5pts) 20 hrs E3 0 hrs E1 10 hrs E2 20 hrs E3 Name_____________________________ page 4 9.) What DNA sequences are important for factor independent transcriptional termination? How are these thought to promote transcription termination? (3pts) 10.) What DNA sequences are important for factor dependent transcriptional termination? How are these thought to promote transcription termination? (3pts) 11.) Briefly describe (or draw) the events involved during translation elongation (do not include events that are associated specifically with initiation or termination). Include the following terms, if appropriate (not all terms are appropriate). TATA Box, 30S Subunit, 50S Subunit, 70S Subunit, RNA Polymerase, Shine Delgarno, rho, ATP, GTP, formylmethionine, P site, A site, RF1, IF3 and IF1, IF2, EF-Tu, EF-G, t-RNA, sigma factor. (6pts) You’ve identified a small gene from a strain of Streptomyces whose expression prevents other bacteria from growing when Streptomyces is present. You are interested in this peptide because of its potential as a new antimicrobial agent. The entire open reading frame (ie. the TRANSLATED portion of the mRNA transcript has following the sequence: 5'… GUG AUC AUU AUA ACU ACC ACA ACG UAA… 3' 12.) Using the table for the genetic code and your knowledge of how translation occurs, translate the given DNA sequence into its appropriate amino acids. (4pts) 13.) What’s the absolute minimum number of different tRNA molecules that would be needed to translate this peptide? Why? (2pts) Name_____________________________ page 5 The gene below encodes Homo sapiens coagulation factor III, a cell surface glycoprotein that enables cells to initiate the blood coagulation cascade. This protein is the only one in the coagulation pathway for which a congenital deficiency has not been described. Your working for a biotech company that is interested in producing drugs to treat specific forms of hemophelia. Your task is to clone this gene into an expression vector as a potential mode of treatment. 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 gggggggggg cccaacctcc gacatggaga ctcctgctcg gcagcatata aaacccgtca aaatgctttt aagcagacgt tctgctgggg ctcggacagc gaagatgaac ggcaaggact gccaaaacaa agtgttcaag gagtgtatgg gtatttgtgg gcaggagtgg tgttggagct tacatggaaa gatatgacct atgtaacgaa tgcacataac caaaaacaaa tttttttttt tctcggctca gagtagctgg gagatggggt ccaccttggc agcttttgag atttctagga ggaatacatt gagacattgg taagtggcat agagtttatg aaaaaggttt atattgagat cccccgcgca ccagccccac cccctgcctg gctgggtctt atttaacttg atcaagtcta acacaacaga acttggcacg agcctctgta caacaattca ggactttagt taatttatac acactaatga cagtgattcc gccaggagaa tcatcatcct ggcagagctg actgcaaatg cgcaaatgag gttattacca tggtactaca atgctttaga tgggaaaatg tttgagacgg cttgcaccct gattacaggt ttcaccatct ctcccaaaga gggctgactt cttttctaac tggaaattca tattctgggc taaacatttg atttaaggta ttctatatgg aatttattta ccccctcgca gggcgccacg gccccgggtc cgcccaggtg gaaatcaact cactgttcaa cacagagtgt ggtcttctcc tgagaactcc gagttttgaa cagaaggaac actttattat gtttttgatt ctcccgaaca aggggaattc tgtcatcatc gaaggagaac ctatattgca tatttcggag ttagcattct accaattcca ttatatattc tcttaaaaaa agtcttgctc ccgtctctcg gcgcactacc tggccaggct tgctagtatt caatccatgt atatgtctat aaacaattgg agcttcctaa agagctaact cttaaagctt ggattttcta atatacttta ctccctctgg gaacccgctc ccgcgccccg gccggcgctt aatttcaaga ataagcacta gacctcaccg tacccggcag ccagagttca caggtgggaa aacactttcc tggaaatctt gatgtggata gttaaccgga agagaaatat ctggctatat tccccactga ctgtgaccga catgaagacc ggttttgaca agttttaatt cgcacttaag tcctgggtgg tgttgcccag ggttcaagca acgccaagct ggtcttgaat atgggcgtga aggaaagtaa aatatagtgt gcaaactttg tatgctttac atatttttat ctatggttga tttatgtagg aataaaggtg ccggcccagg gatctcgccg agaccgccgt caggcactac caattttgga agtcaggaga acgagattgt ggaatgtgga caccttacct caaaagtgaa taagcctccg caagttcagg aaggagaaaa agagtacaga tctacatcat ctctacacaa atgtttcata gaacttttaa ctggagttca tcagcattag tttaacacca gattaaccag acttttgaaa gctggagtgc attgtctgcc aatttttgta tcctgacctc accaccatgc aatggaagga ttaggttctt tattaatgtg aatctgcact aagactacta cattgtatat taatattgtt actggaaaaa gcgccttcag ccaactggta cgctcggacg aaatactgtg gtgggaaccc ttggaaaagc gaaggatgtg gagcaccggt ggagacaaac tgtgaccgta ggatgttttt aaagaaaaca ctactgtttc cagcccggta tggagctgtg gtgtagaaag aaggaagcac gaggatagaa aaaaactctt tcactttgaa tggcaccttt gtcgtccaag agcttttttt agtagcacga tcagcctccc ttttttagta agtgatccac ccagccgaaa aattgggtgc ttttttttca ttaagtgcag ttaactgact tacaaactac ataatttttt ctatttgtat tttttttttt 14.) Design two 15 base primers that you could use in a PCR reaction to amplify this gene from human cells? (4pts) 15.) You know that you will need to clone your PCR product into a BamHI restriction site, GGATCC, found on the expression vector. Modify your primers below such that you will be able to easily clone the PCR product into a BamHI site on an expression vector. (3pts) 16.) The human genome contains 3.3*109 basepairs. Is the 15 base sequence of your primer likely to be unique in the human genome? On average, how many times would your primer Name_____________________________ sequence be found on the human genome? Show your work (4pts) page 6 Name_____________________________ page 7 Your biotech company is using 4 different experimental cloning vectors called pEXPRESS1, pEXPRESS2, pEXPRESS3, and pEXPRESS4. All the vectors have similar constructions (shown to the right). However, the cloning region on each vector is slightly different. Each of the vector cloning regions is shown below. 18.) For each vector, determine if the Coagulation factor III product is likely to be expressed when your PCR product is cloned into the Bam HI site. Explain Why or Why not in each case. (8 pts) Promoter Shine Stop Transcription BamHI site -35-10 Delgarno ATG codons termination GGA TCC consensus sequence UAA UAG sequences Cloning Region of pEXPRESS 1 pEXPRESS1: Shine Promoter Stop Transcription ATG BamHI site codons Delgarno -35-10 termination GGA TCC UAA UAG sequence consensus sequences Cloning Region of pEXPRESS 2 pEXPRESS2: Promoter Shine Stop Transcription -35-10 Delgarno ATG BamHI site codons termination GGA TCC UAA UAG consensus sequence sequences Cloning Region of pEXPRESS 3 pEXPRESS3: Promoter Shine -35-10 Delgarno ATG BamHI site GGA TCC consensus sequence Transcription Stop termination codons sequences UAA UAG Cloning Region of pEXPRESS 4 pEXPRESS4: Cloning Region pEXPRESS Vectors ampicillin resistance origin of replication Name_____________________________ page 8 The Luria-Delbruck experiment demonstrated that bacteria can become resistant to bacteriophage T1 infection through random mutations, rather than through a directed change. You decide to try and see if the random mutation hypothesis also applies to how bacteria become resistant to the antibiotic, rifampicin. You do the following experiment. You inoculate 200 mls of media with a dilute bacterial culture. Then, you split the culture A) growing 100 mls in a single flask, and B) growing the other half in 100 separate, 1 ml test tubes. After allowing the cultures to grow for several hours, you spread A) 1ml aliquots from the single flask on 100 plates that contain rifampicin, and B) the 100 1ml cultures onto 100 plates that contain rifampicin. You allow each set to incubate overnight and count the number of rifampicin resistant colonies on each plate in the morning. Five representative plates from A) the single 100 ml flask are shown below: 19.) If mutations arose primarily through a process of directed change, CLEARLY diagram how you would expect to five representative plates to look from B) the 100 individually grown 1ml cultures. Explain why. (4pts) 20.) If mutations primarily arose randomly within growing populations, CLEARLY diagram how you would expect to five representative plates to look from B) the 100 individually grown 1ml cultures. Explain why. (4pts) 21.) Does this experiment involve positive or negative selection? (2 pts) 23.) If 20 out of your 100 plates of 1ml cultures had no colonies on them, and the cultures had 108 cells/ml, calculate the mutation rate (a) for generating resistence to rifampicin using the Poisson expression, where the probability of having i mutations per culture is represented by P(i) = ( mi e-m )/ i! and a = m/n. (5pts).
